Instruction Manual Parainfluenza 1/2/3 IgM ELISA Enzyme immunoassay based on microtiter plate for the detection and quantitative determination of human IgM antibodies against Parainfluenza 1/2/3 in serum and plasma Cat. No.: 40-375-380064 Storage: 4-8 C For research use only September 2009
Contents Page 1. Intended Use 3 2. General Information 3 3. Principle of the Test 3 4. Limitations, Precautions and General Comments 4 5. Reagents Provided 4 6. Materials Required but not Provided 5 7. Specimen Collection and Handling 6 8. Assay Procedure 6 9. Evaluation 7 10. Assay Characteristics 8 11. References 8 For research use only. 2
1. Intended Use The GenWay Parainfluenza 1/2/3 IgM antibody ELISA kit has been designed for the detection and the quantitative determination of specific IgM antibodies against Parainfluenza 1/2/3 in serum and plasma. Further applications in other body fluids are possible and can be requested from the Technical Service of GenWay. This assay is intended for research use only. 2. General Information The infection with parainfluenza viruses is air-borne from man-to-man. Various species of animals may serve as virus reservoir. Parainfluenza viruses are endemically spread worldwide. The seroprevalence of parainfluenza in infants in their first year of life is 50%. Typical for parainfluenza viruses are frequent reinfections, this applies particularly to parainfluenza 3 viruses. Incubation time is 2-6 days. The parainfluenza viruses are a subgroup of the paramyxoviruses. They are of the same size of approximately 150-300 nm. They are ether-sensitive, agglutinate human or chicken erythrocytes and have a receptor-destructive enzyme, as known from influenza viruses. They can be cultivated best in primary monkey cell cultures or in human epithel cell cultures, however, less successful in embryonized chicken eggs. lt is differentiated between parainfluenza 1, 2, 3 and 4. Together with the respiratory syncytial viruses (RS viruses), the pathogens belong to the major viral pathogens of diseases of the respiratory tract, accompanied by severe symptoms. In adults, parainfluenza virus causes a feverish rhinitis and laryngitis. First signs are sudden headaches, pain in muscles and joints, followed by fever of 38-39 C. If the lower respiratory tract is involved, additionally trachyphonea and dry cough develops as a sign of tracheobronchitis. Parainfluenza 1 causes severe pneumonias in newborns, manifested by high fever, cyanosis, dyspnoea and bloody purulent sputum. Sometimes. meningitis symptoms occur at the same time. Parainfluenza 2 very often causes an acute laryngotracheobronchitis with pseudocroup in infants and children. First signs of the infection are catarrhal symptoms, followed by trachyphoena, dry barking cough and inspiratory stridor. Parainfluenza 3 viruses are considered the major pathogens of pneumonia and bronchiolitis. While types 1, 2 and 3 are distributed worldwide, parainfluenza type 4 appears only in the USA. Infections 1 and 3 occur all the year, while parainfluenza 2 and 4 viruses appear only sporadically. Laboratory diagnosis of parainfluenza viruses is done with haemagglutination inhibiting test (HIT) complement binding reaction (CF) and neutralisation test (NT). Newer methods are IFA and ELISA, which allow to identify IgG and IgA antibodies in the sample serum. In differential diagnosis, tests for other paramyxoviruses like mumps, shipping fever viruses and simianvirus type 5 have to be performed due to possible cross-reactions. 3. Principle of the Test The GenWay Parainfluenza 1/2/3 IgM antibody test kit is based on the principle of the enzyme immunoassay (EIA). Parainfluenza 1/2/3 antigen is bound on the surface of the microtiter strips. Diluted sample serum or ready-to-use standards are pipetted into the wells of the microtiter plate. A binding between the IgM antibodies of the serum and the immobilized Parainfluenza 1/2/3 antigen takes place. After a one hour incubation at room temperature, the plate is rinsed with diluted wash solution, in order to remove unbound material. Then ready-to-use anti-human-igm peroxidase conjugate is added and incubated for 30 minutes. After a further washing step, the substrate (TMB) solution is pipetted and incubated for 20 minutes, inducing the development of a blue dye in the wells. The color development is terminated by the addition of a stop solution, which changes the color from blue to yellow. The resulting dye is measured spectrophotometrically at the wavelength of 450 nm. The concentration of IgM antibodies is directly proportional to the intensity of the color. For research use only. 3
4. Limitations, Precautions and General Comments Only for research use! Do not ingest or swallow! The usual laboratory safety precautions as well as the prohibition of eating, drinking and smoking in the lab have to be followed. All sera and plasma or buffers based upon, have been tested respective to HBsAg, HIV and HCV with recognized methods and were found negative. Nevertheless precautions like the use of latex gloves have to be taken. Serum and reagent spills have to be wiped off with a disinfecting solution (e.g. sodium hypochlorite, 5%) and have to be disposed of properly. All reagents have to be brought to room temperature (18 to 25 C) before performing the test. Before pipetting all reagents should be mixed thoroughly by gentle tilting or swinging. Vigorous shaking with formation of foam should be avoided. It is important to pipet with constant intervals, so that all the wells of the microtiter plate have the same conditions. When removing reagents out of the bottles, care has to be taken that the stoppers are not contaminated. Further a possible mix-up has to be avoided. The content of the bottles is usually sensitive to oxidation, so that they should be opened only for a short time. In order to avoid a carry-over or a cross-contamination, separate disposable pipet tips have to be used. No reagents from different kit lots have to be used, they should not be mixed among one another. All reagents have to be used within the expiry period. In accordance with a Good Laboratory Practice (GLP) or following ISO9001 all laboratory devices employed should be regularly checked regarding the accuracy and precision. This refers amongst others to microliter pipets and washing or reading (ELISA-Reader) instrumentation. The contact of certain reagents, above all the stopping solution and the substrate with skin, eye and mucosa has to be avoided, because possible irritations and acid burns could arise, and there exists a danger of intoxication. 5. Reagents Provided Store kit components at 2-8 o C and do not use after the expiry date on the box outer label. Before use, all components should be allowed to warm up to ambient temperature (18-25 o C). After use, the plate should be resealed, the bottle caps replaced and tightened and the kit stored at 2-8 o C. The opened kit should be used within three months. Components Volume / Qty. Parainfluenza 1/2/3 antigen coated microtiter strips 12 Calibrator A (Negative Control) 2 ml Calibrator B (Cut-Off Standard) 2 ml Calibrator C (Weak Positive Control) 2 ml Calibrator D (Positive Control) 2 ml Enzyme Conjugate 15 ml Substrate 15 ml Stop Solution 15 ml Sample Diluent 60 ml Washing Buffer (10 ) 60 ml Plastic foils 2 Plastic bag 1 For research use only. 4
5.1. Microtiter Strips 12 strips with 8 breakable wells each, coated with a Parainfluenza 1/2/3 antigen (purified mixed extract from Sendai and Greer strains, ATCC VR, of Parainfluenza 1, 2 and 3). Ready-to-use. 5.2. Calibrator A (Negative Control) 2 ml, protein solution diluted with PBS, contains no IgM antibodies against Parainfluenza 1/2/3. Addition of 0.01 % methylisothiazolone and 0.01 % bromonitrodioxane. Ready-to-use. 5.3. Calibrator B (Cut-Off Standard) 2 ml human serum diluted with PBS, contains a low concentration of IgM antibodies against Parainfluenza 1/2/3. Addition of 0.01 % methylisothiazolone and 0.01 % bromonitrodioxane. Ready-to-use. 5.4. Calibrator C (Weak Positive Control) 2 ml, human serum diluted with PBS, contains a medium concentration of IgM antibodies against Parainfluenza 1/2/3. Addition of 0.01 % methylisothiazolone and 0.01 % bromonitrodioxane. Ready-to-use. 5.5. Calibrator D (Positive Control) 2 ml, human serum diluted with PBS, contains a high concentration of IgM antibodies against Parainfluenza 1/2/3. Addition of 0.01 % methylisothiazolone and 0.01 % bromonitrodioxane. Ready-to-use. 5.6. Enzyme Conjugate 15 ml, anti-human-igm-hrp (rabbit), in protein-containing buffer solution. Addition of 0.01 % methylisothiazolone and 0.01 % bromonitrodioxane and 5 mg/l Proclin TM. Ready-to-use. 5.7. Substrate 15 ml, TMB (tetramethylbenzidine). Ready-to-use. 5.8. Stop Solution 15 ml, 0.5 M sulfuric acid. Ready-to-use. 5.9. Sample Diluent 60 ml, PBS/BSA buffer. Addition of 0.095 % sodium azide. Ready-to-use. 5.10. Washing Buffer 60 ml, PBS + Tween 20, 10x concentrate. Final concentration: dilute 1+9 with distilled water. If during the cold storage crystals precipitate, the concentrate should be warmed up at 37 C for 15 minutes. 5.11. Plastic Foils 2 pieces to cover the microtiter strips during the incubation. 5.12. Plastic Bag Resealable, for the dry storage of non-used strips. 6. Materials Required but not Provided 5 µl-, 100 µl- and 500 µl micro- and multichannel pipets Microtiter Plate Reader (450 nm) Microtiter Plate Washer Reagent tubes for the serum dilution Bidistilled water For research use only. 5
7. Specimen Collection and Handling Principally serum or plasma (EDTA, heparin) can be used for the determination. Serum is separated from the blood, which is aseptically drawn by venipuncture, after clotting and centrifugation. The serum or plasma samples can be stored refrigerated (2-8 C) for up to 48 hours, for a longer storage they should be kept at -20 C. The samples should not be frozen and thawed repeatedly. Lipemic, hemolytic or bacterially contaminated samples can cause false positive or false negative results. For the performance of the test the samples (not the standards) have to be diluted 1:101 with readyto-use sample diluent (e.g. 5 µl serum + 500 µl sample diluent). 8. Assay Procedure 8.1. Preparation of Reagents Washing Solution: dilute before use 1+9 with distilled water. If during the cold storage crystals precipitate, the concentrate should be warmed up at 37 C for 15 minutes. Strict adherence to the protocol is advised for reliable performance. Any changes or modifications are the responsibility of the user. All reagents and samples must be brought to room temperature before use, but should not be left at this temperature longer than necessary. Standards and samples should be assayed in duplicates. A standard curve should be established with each assay. Return the unused microtiter strips to the plastic bag and store them dry at 2-8 C. 8.2. Assay Steps 1. Prepare a sufficient amount of microtiter wells for the standards, controls and samples in duplicate as well as for a substrate blank. 2. Pipet 100 µl each of the diluted (1:101) samples and the ready-to-use standards and controls respectively into the wells. Leave one well empty for the substrate blank. 3. Cover plate with the enclosed foil and incubate at room temperature for 60 minutes. 4. Empty the wells of the plate (dump or aspirate) and add 300 µl of diluted washing solution. This procedure is repeated totally three times. Rests of the washing buffer are afterwards removed by gentle tapping of the microtiter plate on a tissue cloth. 5. Pipet 100 µl each of ready-to-use conjugate into the wells. Leave one well empty for the substrate blank. 6. Cover plate with the enclosed foil and incubate at room temperature for 30 minutes. 7. Empty the wells of the plate (dump or aspirate) and add 300 µl of diluted washing solution. This procedure is repeated totally three times. Rests of the washing buffer are afterwards removed by gentle tapping of the microtiter plate on a tissue cloth. 8. Pipet 100 µl each of the ready-to-use substrate into the wells. This time also the substrate blank is pipetted. 9. Cover plate with the enclosed foil and incubate at room temperature for 20 minutes in the dark (e.g. drawer). 10. To terminate the substrate reaction, pipet 100 µl each of the ready-to-use stop solution into the wells. Pipet also the substrate blank. 11. After thorough mixing and wiping the bottom of the plate, perform the reading of the absorption at 450 nm (optionally reference wavelength of 620 nm). The color is stable for at least 60 minutes. For research use only. 6
9. Evaluation The mean values for the measured absorptions are calculated after subtraction of the substrate blank value. The difference between the single values should not exceed 10 %. Example OD Value corrected OD Mean OD Value Substrate Blank 0.014 Negative Control 0.021 / 0.019 0.007 / 0.005 0.006 Cut-Off Standard 0.599 / 0.620 0.585 / 0.606 0.596 Weak Positive Control 1.241 / 1.213 1.227 / 1.199 1.213 Positive Control 2.339 / 2.397 2.325 / 2.383 2.354 The above table contains only an example, which was achieved under arbitrary temperature and environmental conditions. The described data constitute consequently no reference values which have to be found in other laboratories in the same way. 9.1. Qualitative Evaluation The calculated absorptions for the sample sera, as mentioned above, are compared with the value for the cut-off standard. If the value of the sample is higher, there is a positive result. For a value below the cut-off standard, there is a negative result. It seems reasonable to define a range of +/-20 % around the value of the cut-off as a grey zone. In such a case the repetition of the test with the same serum or with a new sample of the same individual, taken after 2-4 weeks, is recommended. Both samples should be measured in parallel in the same run. The positive control must show at least the double absorption compared with the cut-off standard. 9.2. Quantitative Evaluation The ready-to-use standards and controls of the Parainfluenza 1/2/3 antibody kit are defined and expressed in arbitrary units (U/mL). This results in an exact and reproducible quantitative evaluation. Consequently for a given sample follow-up controls become possible. The values for controls and standards in units are printed on the labels of the vials. For a quantitative evaluation the absorptions of the standards and controls are graphically drawn against their concentrations. From the resulting reference curve the concentration values for each sample can then be extracted in relation to their absorptions. It is also possible to use automatic computer programs. For research use only. 7
10. Assay Characteristics Parainfluenza 1/2/3 ELISA Intra-Assay- Precision Inter-Assay- Precision IgG IgA IgM 5.4 % 7.6 % 8.3 % 7.7 % 7.7 % 8.9 % Inter-Lot-Precision 2.9 8.0 % 2.6 10.4 % 4.3 8.3 % Analytical Sensitivity 1.04 U/mL 0.96 U/ml 1.18 U/mL Recovery 96 106 % 94 106 % 87 104 % Linearity 82 110 % 79 105 % 67 126 % Cross-Reactivity Interferences No cross-reactivity to RSV, Adenovirus and Bordetella. No interferences to bilirubin up to 0.3 mg/ml, hemoglobin up to 8.0 mg/ml and triglycerides up to 5.0 mg/ml Clinical Specificity 100 % 99 % 100 % Clinical Sensitivity 100 % 100 % 100 % 11. References 1. Collins PL, Chanock RM, McIntosh K: Parainfluenza viruses, in Fields BN (ed): Fields Virology. Philadelphia, Lippincott-Raven Publishers, 1996, pp 1205-1241. 2. Chanock RM: Association of a new type of cytopathogenic myxovirus with infantile croup. J Exp Med 104:555-576, 1956. 3. Andrewes CH, Bang FB, Chanock RM, et al: Parainfluenza viruses 1, 2, and 3: Suggested names for recently described myxoviruses. Virology 8:129-130, 1959. 4. Hotez PJ, Doveikis SA: Adult respiratory distress syndrome associated with parainfluenza virus type 1 in children. Pediatr Infect Dis J 9:750-752, 1990. 5. Monto AS: The Tecumseh study of respiratory illness: V: Patterns of infection with the parainfluenza viruses. Am J Epidemiol 97:338-348, 1973. 6. Hornsleth A: Respiratory virus disease in infancy and childhood in Copenhagen 1963-65: an estimation of the etiology based on complement fixation tests. Acta Pathol Microbiol Scand 69:287-303, 1967. 7. Killgore GE, Dowdle WR: Antigenic characterization of parainfluenza 4a and 4b by the hemagglutination-inhibition test and distribution of HI antibody in human sera. Am J Epidemiol 91:308-316, 1970. 8. LaPlaca M, Moscovici C: Distribution of parainfluenza antibodies in different groups of population. J Immunol 88:72-77, 1962. 9..Fan J, Henrickson KJ: Rapid diagnosis of human parainfluenza virus type 1 infection by quantitative reverse transcription-pcr-enzyme hybridization assay. J Clin Microbiol 34:1914-1917, 1996. 10. Karron RA, Wright PF, Hall SL, et al: Live attenuated bovine parainfluenza virus type 3 vaccine is safe, infectious, immunogenic, and phenotypically stable in infants and children. J Infect Dis 171:1107-1114, 1995. For research use only. 8